The present invention provides methods and compositions for the identification and use of IKK inhibitors. In particular, the present invention provides cyclopentenone prostaglandins suitable for use as IKK inhibitors.
The rapid and precise control of gene expression via transcription factors is critical to the survival of cells. Depending upon the inducing stimulus, it can be critical to the survival of a cell to have one or more genes rapidly induced, so that the resultant products are active. For example, an inflammatory response stimulated by an injury or infection, results in rapid vasodilation in the injured area and infiltration of effector cells such as macrophages. Vasodilation occurs within seconds or minutes of the response and is due, in part, to the expression of cytokines by cells in the injured region.
The rapid induction of the inflammatory and immune responses requires that the transcription factors involved in regulating such responses be present in the cell in a form that is amenable to rapid activation. Thus, upon exposure to an inducing stimulus, the response can quickly occur. If such transcription factors are not present in a cell in an inactive state, it is necessary to synthesize the factors upon exposure to an inducing stimulus, greatly reducing the speed with which a response can occur.
Regulation of transcription factor activities involved in such rapid gene induction can occur by various mechanisms. For example, in some cases, a transcription factor that exists in an inactive state in a cell can be activated by post-translational modification (e.g., phosphorylation of one or more serine, threonine, or tyrosine residues). Furthermore, the transcription factor can be rendered inactive by association of the factor with a regulatory factor, which, upon exposure to an inducing stimulus, is released from the transcription factor, thereby activating the transcription factor. Alternatively, an inactive transcription factor may have to associate with a second protein in order to produce transcriptional activity.
Rarely, as in the case of glucocorticoids, the inducing stimulus directly interacts with the inactive transcription factor, rendering it active and resulting in the induction of gene expression. However, more often, an inducing stimulus initiates the induced response by interacting with a specific receptor present on the cell membrane or by entering the cell and interacting with an intracellular protein. Furthermore, the signal generally is transmitted along a pathway, for example, from the cell membrane to the nucleus, due to a series of protein interactions. Such signal transduction pathways allow for the rapid transmission of extracellular inducing stimuli, such that appropriate gene expression is rapidly induced.
Although the existence of signal transduction pathways has long been recognized and many of the cellular factors involved in such pathways have been described, the pathways responsible for the expression of many critical responses, including the inflammatory and immune responses, have only been incompletely defined. For example, it is recognized that various inducing stimuli such as viruses and bacteria, activate common arms of the immune and inflammatory response. However, differences in the gene products expressed also are observed, indicating that these stimuli share certain signal transduction pathways, but also induce other pathways unique to the inducing stimulus. Furthermore, since inducing agents such as bacteria and viruses initially stimulate different signal transduction pathways, yet induce the expression of common genes, some signal transduction pathways must converge at a point such that the different pathways activate common transcription factors.
A clearer understanding of the proteins involved in such pathways facilitates descriptions of drug mechanisms of action. For example, such an understanding facilitates the determination of a drug""s mechanism of action (e.g., in cases where the drug is known to interfere with gene expression regulated by a particular pathway, but the target of which is unknown). In addition, an understanding of the pathways involved facilitates the identification of defect(s) in the pathway(s) associated with diseases such as cancer. For example, the altered expression of cell adhesion molecules is associated with the ability of a cancer cell to metastasize. However, the critical proteins involved in the signal transduction pathway leading to expression of cell adhesion molecules have not been identified. Thus, there is a need in the art to identify the proteins involved in signal transduction pathways, particularly those proteins that result in the induction of gene products involved in the inflammatory and immune responses. Indeed, despite much research in the field, there remains a need in the art for compositions and methods for treating and/or preventing infectious as well as other diseases, and inflammation.
The present invention provides methods for identifying compounds with activities that inhibit IKK. In some preferred embodiments, the methods comprise contacting IKK, or a subunit or fragment of IKK with the compound. In some particularly preferred embodiments, the compound interacts with cysteine-179 of the IKK. In alternative embodiments, the method further comprises determining whether the compound interacts with cysteine-179, wherein the interaction with cysteine-179 correlates with activity as an IKK inhibitor.
The present invention also provides methods to prevent and/or treat diseases and/or conditions associated with NFxcexaB. In particularly preferred embodiments, the method involves inhibition of the action of NFxcexaB. The present invention further provides methods for the inhibition of IKK in vitro and in vivo, as well as methods to prevent NFxcexaB activation in an animal. In some preferred embodiments, the animal is a mammal. In particularly preferred embodiments, the mammal is a human.
The present invention also provides methods to improve the therapeutic activity of COX2 inhibitors in animals (e.g., mammals, including but not limited to humans), comprising administering the COX2 inhibitor in combination with a compound that inhibits IKK activity to an animal. It is contemplated that COX2 inhibitors such as celecoxib, or Fecoxib, NS-398, and PD 98059 (Jones et al., Nat. Med., 18:1418-1423 [1999]), will find use with the present invention. The present invention further provides methods to inhibit the activity of IKK in a cell, comprising contacting the cell with a compound that interacts with IKK. In particularly preferred embodiments, the compound interacts with cysteine-179 of IKK.
The present invention further provides compounds, as well as pharmaceutical compositions comprising compounds that inhibit IKK. The present invention also provides pharmaceutical compositions comprising compounds that are COX2 inhibitors. In additional embodiments, the present invention provides pharmaceutical compositions comprising at least one compound that inhibits IKK, a COX2 inhibitor, and a pharmaceutically accepted carrier.
The present invention also provides the use of the compound of formula I, wherein R1 and R2 are each independently selected from hydrogen, (C1-C12)alkyl, and (C2-C12)alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or more hydroxy, halo, nitro, trifluoromethyl, cyano, NRR, SR, or COOR; each R is independently selected from the group consisting of hydrogen and (C1-C12)alkyl; wherein the optional bond (i.e., the xe2x80x9c - - - xe2x80x9d bond) is present or absent, provided that when the optional bond is present, R1 is not hydrogen; and n is 0, 1, 2, or 3; or a pharmaceutically acceptable salt thereof.
The present invention further provides methods for the preparation of medicaments for inhibiting IKK and/or NFxcexaB in an animal, and/or for improving the therapeutic activity of a COX2 inhibitor in an animal. In some preferred embodiments, the present invention provides the use of combination therapies. In these embodiments, at least one compound that inhibits IKK and at least one compound that inhibits COX2 are used in combination. In some embodiments, one compound exhibits multiple activities (e.g., the compound inhibits COX2, as well as IKK and/or NFxcexaB, or any combination of these activities). In still further embodiments, the present invention provides methods to prevent NFxcexaB activation in an animal, comprising inhibiting IKK activity in the animal. In particularly preferred embodiments, IKK activity is inhibited by the administration of a compound that interacts with cysteine-179 of IKK, in an amount effective for the inhibition of IKK activity.
The present invention provides methods for the inhibition of IKK, comprising contacting IKK with a compound that interacts with the cysteine at position 179 of IKK. In some embodiments, the contacting is in vitro, while in other embodiments, it is in vivo. In some particularly preferred embodiments, the compound interacts by forming a covalent bond with the cysteine at position 179 of IKK. In other preferred embodiments, the compound is a Michael acceptor. In further preferred embodiments, the compound is selected from the group consisting of cyclopentenones and substituted cyclopentenones. In some particularly preferred embodiments, the compound is a cyclopentenone prostaglandin. In still further preferred embodiments, the compound is selected from the group consisting of xcex1,xcex2-unsaturated ketones, xcex1,xcex2-unsaturated esters, and xcex1,xcex2-unsaturated nitrites. In some alternatively preferred embodiments, the compound is a compound of formula I: 
wherein R1 and R2 are each independently selected from hydrogen, (C1-C12)alkyl, and (C2-C12)alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or more hydroxy, halo, nitro, trifluoromethyl, cyano, NRR, SR, or COOR; each R is independently selected from the group consisting of hydrogen and (C1-C12)alkyl; wherein the optional bond is present or absent, provided that when the optional bond (indicated by xe2x80x9c - - - xe2x80x9d) is present, R, is not hydrogen; and n is 0, 1, 2, or 3; or a pharmaceutically acceptable salt thereof. In still further particularly preferred embodiments. the compound is selected from the group consisting of PGA1, 15-deoxy-xcex9412-14PGJ2, and pharmaceutically acceptable salts thereof.
The present invention also provides methods for identifying a compound with activity as an inhibitor of IKK, comprising: providing an IKK, wherein the IKK is selected from the group consisting of full-length IKK, subunits of IKK, and fragments of IKK, wherein the IKK comprises a cysteine at position 179, and a test compound; exposing the IKK to the test compound; and determining whether the compound interacts with the cysteine at position 179 of IKK. In some preferred embodiments, the interaction with the compound and the cysteine at position 179 of IKK comprises inhibiting IKK. In some particularly preferred embodiments, the compound interacts with the cysteine at position 179 of IKK by forming a covalent bond with the cysteine at position 179 of IKK.
The present invention further provides methods to improve the therapeutic activity of a COX2 inhibitor in animal, comprising administering a COX2 inhibitor in combination with a compound that inhibits IKK activity to an animal. In some embodiments, the compound is a Michael acceptor. In alternative embodiments, the compound is selected from the group consisting of cyclopentenones and substituted cyclopentenones. In some particularly preferred embodiments, the compound is a cyclopentenone prostaglandin. In further preferred embodiments, the compound is an xcex1,xcex2-unsaturated carbonyl compound. In additional particularly preferred embodiments, the compound is selected from the group consisting of xcex1,xcex2-unsaturated ketones, xcex1,xcex2-unsaturated esters, and xcex1,xcex2-unsaturated nitrites. In still further particularly preferred embodiments, the compound is a compound of formula I: 
wherein R1 and R2 are each independently selected from hydrogen, (C1-C12)alkyl, and (C2-C12)alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or more hydroxy, halo, nitro, trifluoromethyl, cyano, NRR, SR, or COOR; each R is independently selected from the group consisting of hydrogen and (C1-C12)alkyl; wherein the optional bond (indicated by xe2x80x9c - - - xe2x80x9d) is present or absent, provided that when the optional bond is present, R1 is not hydrogen; and n is 0, 1, 2, or 3; or a pharmaceutically acceptable salt thereof. In still other particularly preferred embodiments, the compound is selected from the group consisting of PGA1, 15-deoxy-xcex9412-14, and pharmaceutically acceptable salts thereof.
The present invention also provides methods to prevent disease associated with NFxcexaB, comprising: providing an animal at risk for disease associated with NFxcexaB, wherein the animal expresses IKK, and a composition that inhibits IKK; and administering the compound to the animal under conditions such that the composition inhibits IKK expressed by the animal, resulting in inhibition of NFxcexaB. In some preferred embodiments, the animal is a human. In additional embodiments, the composition comprises a compound that interacts with cysteine-179 of IKK. In still further embodiments, the disease is selected from the group consisting of inflammation, cancer, bacterial, and viral diseases. In some preferred embodiments, the composition comprises at least one cyclopentenone. In some particularly preferred embodiments, the cyclopentenone is a cyclopentenone prostaglandin.
The present invention further provides methods for the inhibition of IKK, comprising contacting the IKK with a composition that interacts with cysteine-179 of IKK. In some embodiments, the composition interacts with IKK forms a covalent bond with the cysteine-179 of IKK. However, it is not intended that the present invention be limited to this particular type of interaction. In some preferred embodiments, inhibition of IKK results in prevention of NFxcexaB activation.
The present invention also provides methods to treat disease associated with NFxcexaB, comprising: providing an animal having at least one disease associated with NFxcexaB, wherein the animal expresses IKK, and a composition that inhibits IKK; and administering the compound to the animal under conditions such that the composition inhibits IKK expressed by the animal, resulting in inhibition of NFxcexaB. In some preferred embodiments, the animal is a human. In additional preferred embodiments, the composition comprises a compound that interacts with cysteine-179 of IKK. In still further embodiments, the disease is selected from the group consisting of inflammation, cancer, bacterial, and viral diseases. In some preferred embodiments, the composition comprises at least one cyclopentenone. In some particularly preferred embodiments, the cyclopentenone is a cyclopentenone prostaglandin.